CN104017570A - Preparation method of fluorescent probe for identification of microfilament bacteria - Google Patents
Preparation method of fluorescent probe for identification of microfilament bacteria Download PDFInfo
- Publication number
- CN104017570A CN104017570A CN201410225878.5A CN201410225878A CN104017570A CN 104017570 A CN104017570 A CN 104017570A CN 201410225878 A CN201410225878 A CN 201410225878A CN 104017570 A CN104017570 A CN 104017570A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- preparation
- microfilament
- picoline
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 62
- 241000894006 Bacteria Species 0.000 title claims abstract description 47
- 102000002151 Microfilament Proteins Human genes 0.000 title claims abstract description 47
- 108010040897 Microfilament Proteins Proteins 0.000 title claims abstract description 47
- 210000003632 microfilament Anatomy 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- FKNQCJSGGFJEIZ-UHFFFAOYSA-N 4-methylpyridine Chemical group CC1=CC=NC=C1 FKNQCJSGGFJEIZ-UHFFFAOYSA-N 0.000 claims abstract description 15
- -1 carbon chain compound Chemical class 0.000 claims abstract description 15
- 239000000975 dye Substances 0.000 claims abstract description 13
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims abstract description 12
- 230000001360 synchronised effect Effects 0.000 claims abstract description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 35
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 claims description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 15
- 125000001475 halogen functional group Chemical group 0.000 claims description 13
- 238000004821 distillation Methods 0.000 claims description 12
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- PAQYNMHBAJPURI-UHFFFAOYSA-N 9H-carbazole pyridine Chemical compound C1=CC=NC=C1.C1=CC=C2C3=CC=CC=C3NC2=C1 PAQYNMHBAJPURI-UHFFFAOYSA-N 0.000 claims description 10
- 239000012065 filter cake Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 7
- PLAZXGNBGZYJSA-UHFFFAOYSA-N 9-ethylcarbazole Chemical compound C1=CC=C2N(CC)C3=CC=CC=C3C2=C1 PLAZXGNBGZYJSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000013019 agitation Methods 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000005457 ice water Substances 0.000 claims description 6
- 229910052740 iodine Inorganic materials 0.000 claims description 6
- 239000011630 iodine Substances 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- RZJRJXONCZWCBN-UHFFFAOYSA-N octadecane Chemical compound CCCCCCCCCCCCCCCCCC RZJRJXONCZWCBN-UHFFFAOYSA-N 0.000 claims description 6
- 150000003053 piperidines Chemical class 0.000 claims description 6
- 238000000967 suction filtration Methods 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 5
- SODQFLRLAOALCF-UHFFFAOYSA-N 1lambda3-bromacyclohexa-1,3,5-triene Chemical compound Br1=CC=CC=C1 SODQFLRLAOALCF-UHFFFAOYSA-N 0.000 claims description 3
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 3
- 230000008025 crystallization Effects 0.000 claims description 3
- 229940038384 octadecane Drugs 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- PBLNBZIONSLZBU-UHFFFAOYSA-N 1-bromododecane Chemical compound CCCCCCCCCCCCBr PBLNBZIONSLZBU-UHFFFAOYSA-N 0.000 claims description 2
- WSULSMOGMLRGKU-UHFFFAOYSA-N 1-bromooctadecane Chemical compound CCCCCCCCCCCCCCCCCCBr WSULSMOGMLRGKU-UHFFFAOYSA-N 0.000 claims description 2
- KOFZTCSTGIWCQG-UHFFFAOYSA-N 1-bromotetradecane Chemical compound CCCCCCCCCCCCCCBr KOFZTCSTGIWCQG-UHFFFAOYSA-N 0.000 claims description 2
- KMWHQYDMBYABKL-UHFFFAOYSA-N 1-iodohexadecane Chemical compound CCCCCCCCCCCCCCCCI KMWHQYDMBYABKL-UHFFFAOYSA-N 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 22
- 238000012986 modification Methods 0.000 abstract description 4
- 230000004048 modification Effects 0.000 abstract description 4
- 230000005661 hydrophobic surface Effects 0.000 abstract description 3
- LRAMRYMTIJHJSU-UHFFFAOYSA-N C=CC1=CC=CC=C1.N1=CC=CC=C1.C1=CC=CC=2C3=CC=CC=C3NC12 Chemical compound C=CC1=CC=CC=C1.N1=CC=CC=C1.C1=CC=CC=2C3=CC=CC=C3NC12 LRAMRYMTIJHJSU-UHFFFAOYSA-N 0.000 abstract 2
- QGJXVBICNCIWEL-UHFFFAOYSA-N 9-ethylcarbazole-3-carbaldehyde Chemical compound O=CC1=CC=C2N(CC)C3=CC=CC=C3C2=C1 QGJXVBICNCIWEL-UHFFFAOYSA-N 0.000 abstract 1
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 40
- 239000000047 product Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 239000010802 sludge Substances 0.000 description 8
- 230000005284 excitation Effects 0.000 description 7
- 230000003595 spectral effect Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 5
- 229910052794 bromium Inorganic materials 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- XLTVNRPYGIVKKX-UHFFFAOYSA-N CCCCCCCCCCCCCCCCN1C=CC(C)=CC1.Br Chemical compound CCCCCCCCCCCCCCCCN1C=CC(C)=CC1.Br XLTVNRPYGIVKKX-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 229960000935 dehydrated alcohol Drugs 0.000 description 3
- 238000001953 recrystallisation Methods 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- GSGKOPXYSRKSRW-UHFFFAOYSA-N CCCCCCCCCCCCCCN1C=CC(C)=CC1.Br Chemical compound CCCCCCCCCCCCCCN1C=CC(C)=CC1.Br GSGKOPXYSRKSRW-UHFFFAOYSA-N 0.000 description 2
- 241000589651 Zoogloea Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- WXKPKMSCKIKZID-UHFFFAOYSA-N 1-dodecyl-4-methyl-2H-pyridine Chemical compound C(CCCCCCCCCCC)N1CC=C(C=C1)C WXKPKMSCKIKZID-UHFFFAOYSA-N 0.000 description 1
- LRVPHLQXCIOXRL-UHFFFAOYSA-N 1-hexadecyl-4-methyl-2H-pyridine Chemical compound C(CCCCCCCCCCCCCCC)N1CC=C(C=C1)C LRVPHLQXCIOXRL-UHFFFAOYSA-N 0.000 description 1
- HFKJDLDBOSQYNQ-UHFFFAOYSA-N 4-methyl-1-octadecyl-2H-pyridine Chemical compound C(CCCCCCCCCCCCCCCCC)N1CC=C(C=C1)C HFKJDLDBOSQYNQ-UHFFFAOYSA-N 0.000 description 1
- OXYVKEOVVZEZGP-UHFFFAOYSA-N CCCCCCCCCCCCCCCCCCN1C=CC(C)=CC1.Br Chemical compound CCCCCCCCCCCCCCCCCCN1C=CC(C)=CC1.Br OXYVKEOVVZEZGP-UHFFFAOYSA-N 0.000 description 1
- XXIYGVCQDGSXAZ-UHFFFAOYSA-N CCCCCCCCCCCCN1C=CC(C)=CC1.Br Chemical compound CCCCCCCCCCCCN1C=CC(C)=CC1.Br XXIYGVCQDGSXAZ-UHFFFAOYSA-N 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Indole Compounds (AREA)
- Pyridine Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Plural Heterocyclic Compounds (AREA)
Abstract
The invention provides a preparation method of a fluorescent probe for identification of microfilament bacteria. The method first uses halogenated long carbon chain compound to modify 4-methyl pyridine part of a carbazole pyridine styrene cyanine dye, and then the dye reacts with 3-formyl-N-ethyl carbazole for the synthesis of the fluorescent probe with a long hydrophobic chain. The invention has the beneficial effect that modification and preparation of the carbazole pyridine styrene cyanine dye are synchronized in the probe preparation process, the preparation method is simple, and the prepared fluorescent probe has little background interference, high fluorescence intensity and stable fluorescent property. The characteristic of hydrophobic surface of microfilament bacteria is utilized that the prepared fluorescent probe with long hydrophobic chain in this application can combined with microfilament bacteria to achieve the purpose of fluorescent identification of microfilament bacteria.
Description
Technical field
The invention belongs to the preparation method with fluorescent probe for sewage treatment area microorganism detection, be specifically related to the long carbon chain compound modification of a kind of halo carbazole pyridine styrenic cyanine dyes and obtain identifying the preparation method of fluorescent probe for microfilament bacterium.
Background technology
From activated sludge process, be born more than one since century, sludge bulking problem is just a global problem of the normal operation of puzzlement sewage work always.The filamentous bulking that activated sludge bulking is caused by thread fungus hypertrophy and non-filamentous bulking, wherein the former accounts for leadingly, it is reported and approximately has 90% sludge bulking problem hyphomycetic raised growth and causing in active sludge.People concentrate on 2 aspects to the research of filamentous bulking and crucial flora thereof: the one, cause the research of the crucial flora of filamentous bulking itself, and comprise the identification of the thread flora of being correlated with in its isolation identification, physiological and ecological characteristic and sludge system and quantitatively characterizing etc.; It two is to inquire into the impact of process conditions on thread flora from technological operation angle, seeks to control the countermeasure of sludge bulking.
Microfilament bacterium is modal a kind of thread fungus in mud foaming and expansion process, particularly relates in the Sewage treatment systems of nutrient removal, thereby this class thread fungus is carried out to Study on Identification, significant to the early warning of sludge bulking phenomenon and control.Tradition discrimination method is mainly to carry out according to the morphological feature of microfilament bacterium and staining reaction state.But due to the more difficult separation of microfilament bacterium and pure culture, people understand less to its metabolism and physiological and ecological characteristic.In recent years, along with developing rapidly of the associated molecule biology techniques such as PCR, nucleic acid sequencing, adopt fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) technology is differentiated microfilament bacterium, the limitation of having avoided traditional method to identify and count, and cellular form is not destroyed in mensuration process, can truly reflect the features such as the situation of microorganism under physical environment and distribution.But FISH can only identify the microorganism of known nucleic acid sequence, and there is lower, the low rRNA content of cell-penetrating rate and have the impact of microorganism autofluorescence and the fluorescence shortcoming such as fade in FISH method.
The people such as J.L.Nielsen adopt fluorescent microsphere method to detect microfilament bacterium, find that microfilament bacterium surface has certain hydrophobicity.Utilize its this characteristic, in the application, adopt long carbon chain compound to modify fluorescence dye, build a kind of fluorescent probe with long hydrophobic chain, it is combined with microfilament bacterium, thereby reach the object of fluorescence identification microfilament bacterium.
Summary of the invention
The object of the invention is to provide a kind of preparation method of fluorescent probe for microfilament bacterium that identifies, utilize the long carbon chain compound of halo to modify fluorescence dye, make the fluorescent probe making there is long hydrophobic chain, can be combined with the microfilament bacterium with hydrophobic surface, thereby reach the object of identifying microfilament bacterium.
For achieving the above object; the technical solution used in the present invention is to provide a kind of preparation method of fluorescent probe for microfilament bacterium that identifies; the method is take the synthetic of carbazole pyridine styrenic cyanine dyes and modify synchronized synthetic route; first introducing the long carbon chain compound of halo partly modifies its 4-picoline; then again with 3-formyl radical-N-ethyl carbazole partial reaction; make the fluorescent probe with long hydrophobic chain, comprise the following steps:
(1) chain alkyl-4-picoline halide salt is synthetic
In round-bottomed flask, add respectively 0.8-1.5 part 4-picoline, the long carbon chain compound of 0.7-1.3 part halo and 28 parts of toluene, magnetic agitation is fully dissolved and is reacted, temperature of reaction is 120 ℃, after question response completes, is cooled to room temperature, remove upper toluene and obtain oily matter, and with normal hexane washing, finally the oily matter having washed is carried out in Rotary Evaporators to underpressure distillation, obtain chain alkyl-4-picoline halide salt;
(2) 3-formyl radical-N-ethyl carbazole is synthetic
In round-bottomed flask, add 20 parts of phosphorus oxychloride, under ice-water bath cooling and stirring condition, in round-bottomed flask, drip successively DMF19-22 part, 65 parts containing 1 of 3-3.5 part N-ethyl carbazole, 2-dichloroethane solution, drips and finishes, back flow reaction 8h, reaction solution is cooled to room temperature, and pour in frozen water, stir, with dichloromethane extraction, in Rotary Evaporators, carry out after underpressure distillation, through column chromatography for separation, obtain 3-formyl radical-N-ethyl carbazole;
(3) fluorescent probe is synthetic
Chain alkyl-4-picoline halide salt of preparation in step (1) is dissolved in 70 parts of dehydrated alcohols; and add 3-formyl radical-N-ethyl carbazole of preparation in 0.9-1.3 part step (2); drip 3-5 and drip piperidines, back flow reaction 12h, obtains red solution; after reaction solution is concentrated; add ether to separate out precipitation, suction filtration, makes filter cake; by filter cake crystallization again in methyl alcohol, must there is the fluorescent probe of long hydrophobic chain.
Effect of the present invention is:
(1) prepared fluorescent probe; in the synthetic route of carbazole pyridine styrenic cyanine dyes; first introducing the long carbon chain compound of halo partly modifies its 4-picoline; then again with 3-formyl radical-N-ethyl carbazole partial reaction; thereby prepare the fluorescent probe that the long carbochain of halo is modified; the synchronization that this preparation method has realized the synthetic of carbazole pyridine styrenic cyanine dyes and modified; and this preparation method is simple; the fluorescent probe background interference making is little; fluorescence intensity is high, and photoluminescent property is stable.
(2) modification of the long carbon chain compound of halo makes the fluorescent probe of preparation have long hydrophobic chain, can be combined with the microfilament bacterium with hydrophobic surface, thereby reach the object that fluorescence is identified microfilament bacterium.
Accompanying drawing explanation
Fig. 1 is the microscopy figure of interactively under fluorescent probe of the present invention and microfilament bacterium fluorescence;
Fig. 2 is the microscopy figure of interactively under fluorescent probe of the present invention and microfilament bacterium natural light.
Embodiment
In conjunction with example, identification microfilament bacterium of the present invention is illustrated by the preparation method of fluorescent probe.
The preparation method of fluorescent probe for identification microfilament bacterium of the present invention; realized the preparation of fluorescence dye and modified synchronization; in the synthetic route of carbazole pyridine styrenic cyanine dyes; first introducing the long carbon chain compound of halo partly modifies its 4-picoline; then again with 3-formyl radical-N-ethyl carbazole partial reaction; make the fluorescent probe with long hydrophobic chain, this process mainly comprises the following steps:
(1) chain alkyl-4-picoline halide salt is synthetic
In round-bottomed flask, add respectively 0.8-1.5 part 4-picoline, the long carbon chain compound of 0.7-1.3 part halo and 28 parts of toluene, magnetic agitation is fully dissolved and is reacted, temperature of reaction is 120 ℃, after question response completes, is cooled to room temperature, remove upper toluene and obtain oily matter, and with normal hexane washing, finally the oily matter having washed is carried out in Rotary Evaporators to underpressure distillation, obtain chain alkyl-4-picoline halide salt;
(2) 3-formyl radical-N-ethyl carbazole is synthetic
In round-bottomed flask, add 20 parts of phosphorus oxychloride, under ice-water bath cooling and stirring condition, in round-bottomed flask, drip successively DMF19-22 part, 65 parts containing 1 of 3-3.5 part N-ethyl carbazole, 2-dichloroethane solution, drips and finishes, back flow reaction 8h, reaction solution is cooled to room temperature, and pour in frozen water, stir, with dichloromethane extraction, in Rotary Evaporators, carry out after underpressure distillation, through column chromatography for separation, obtain 3-formyl radical-N-ethyl carbazole;
(3) fluorescent probe is synthetic
Chain alkyl-4-picoline halide salt of preparation in step (1) is dissolved in 70 parts of dehydrated alcohols; and add 3-formyl radical-N-ethyl carbazole of preparation in 0.9-1.3 part step (2); drip 3-5 and drip piperidines, back flow reaction 12h, obtains red solution; after reaction solution is concentrated; add ether to separate out precipitation, suction filtration, makes filter cake; by filter cake crystallization again in methyl alcohol, must there is the fluorescent probe of long hydrophobic chain.
Prepared have the fluorescent probe of long hydrophobic chain and the mechanism of microfilament bacterium is as follows: get the fluorescent probe making and be configured to probe solution, then with the bacterial solution containing microfilament bacterium, probe solution is diluted, probe solution after dilution is dropped on slide glass, adopt fluorescence inverted microscope under fluorescence and natural light condition, to carry out paired observation respectively, respectively as depicted in figs. 1 and 2.Contrast can find out, in Fig. 1, the brightness of microfilament bacterium mycelia is obviously better than Fig. 2, illustrates that the prepared fluorescent probe with long hydrophobic chain of the application can identify microfilament bacterium well.
The long carbon chain compound of described halo is 1-bromo-dodecane, 1-bromo-tetradecane, 1-bromine n-Hexadecane, 1-bromo-octadecane, 1-iodine dodecane, Cetyl Iodide or 1-iodine octadecane.
Embodiment:
Embodiment 1
In round-bottomed flask, add respectively 0.4650g4-picoline, 1.5268g1-bromine n-Hexadecane and 15mL toluene, magnetic agitation is fully dissolved, 120 ℃ of backflows, after having reacted, are cooled to room temperature, remove upper toluene, with normal hexane washing, remain yellow oil three times, the underpressure distillation in Rotary Evaporators of the product after washing is removed to solvent and obtain 1-hexadecyl-4-picoline Bromide, productive rate 91.5%.In round-bottomed flask, add 9.5mL phosphorus oxychloride, under ice-water bath cooling and stirring, drip 7.7mL DMF, drip and finish, in stirring at room 30min, slowly drip 16ml and contain 1 of 3.15g N-ethyl carbazole, 2-dichloroethane solution, drips and finishes, back flow reaction 8h.After having reacted, be cooled to after room temperature; reaction solution is poured in frozen water and stirred 1h, with dichloromethane extraction frozen water solution, then with P2O5, be dried the dichloromethane solution that contains product; methylene dichloride is removed in underpressure distillation afterwards, and product obtains 3-formyl radical-N-ethyl carbazole through column chromatography for separation.In the reaction flask of the 1-hexadecyl-4-picoline Bromide obtaining; add 1.1155g3-formyl radical-N-ethyl carbazole; dehydrated alcohol 20.00mL, drips 3 piperidines, and back flow reaction 12h obtains red solution; after reaction solution is concentrated; add diethyl ether and separate out solid, suction filtration, filter cake is recrystallization in methyl alcohol; obtain required fluorescent probe, productive rate 49.6%.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 435nm, and emission wavelength is 574nm.
Take the fluorescent probe 30.1mg of the 1-bromine n-Hexadecane modification of preparation, be dissolved in 50mL ethanol, be mixed with the probe solution that concentration is 1mmol/L, thereby adopt bacterial solution to dilute and be configured to the probe solution that concentration is 0.010mmol/L it, adopt fluorescence inverted microscope under natural light and fluorescence condition, to carry out paired observation respectively, can obviously find out, under fluorescence condition, detect, can be observed bright microfilament bacterium mycelia, and be obviously different from zoogloea, as shown in Figure 1.And detect under natural light condition, microfilament bacterium mycelia is display brightness and be difficult for being different from zoogloea not, as shown in Figure 2.Comparative illustration, fluorescent probe prepared by the application can be combined with microfilament bacterium, thereby reaches the object of identification microfilament bacterium.
Embodiment 2
In round-bottomed flask, add respectively 0.6985g4-picoline, 1.8023g1-bromo-tetradecane and 15mL toluene, magnetic agitation is fully dissolved, 120 ℃ of backflows, after having reacted, are cooled to room temperature, remove upper toluene, with normal hexane washing, remain yellow oil three times, the underpressure distillation in Rotary Evaporators of the product after washing is removed to solvent and obtain 1-tetradecyl-4-picoline Bromide, productive rate 92.7%.In round-bottomed flask, add 9.5mL phosphorus oxychloride, under ice-water bath cooling and stirring, drip 8.9mLDMF, drip and finish, in stirring at room 30min, slowly drip 25ml and contain 1 of 3.445g N-ethyl carbazole, 2-dichloroethane solution, drips and finishes, back flow reaction 8h.After having reacted, be cooled to after room temperature; reaction solution is poured in frozen water and stirred 1h, with dichloromethane extraction frozen water solution, then with P2O5, be dried the dichloromethane solution that contains product; methylene dichloride is removed in underpressure distillation afterwards, and product obtains 3-formyl radical-N-ethyl carbazole through column chromatography for separation.In the reaction flask of the 1-tetradecyl-4-picoline Bromide obtaining; add 1.45g3-formyl radical-N-ethyl carbazole; dehydrated alcohol 20.00mL, drips 5 piperidines, and back flow reaction 12h obtains red solution; after reaction solution is concentrated; add diethyl ether and separate out precipitation, suction filtration, filter cake is recrystallization in methyl alcohol; obtain required fluorescent probe, productive rate 31%.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 438nm, and emission wavelength is 572nm.As in Example 1, prepared fluorescent probe is configured to the probe solution that concentration is 0.015mmol/L, adopts inverted microscope to observe, can be observed bright microfilament bacterium mycelia under fluorescence condition, prepared probe can be identified microfilament bacterium.
Embodiment 3
In round-bottomed flask, add respectively 0.3725g4-picoline, 0.8723g1-bromo-dodecane and 15mL toluene, magnetic agitation is fully dissolved, 120 ℃ of backflows, after having reacted, are cooled to room temperature, remove upper toluene, with normal hexane washing, remain yellow oil three times, the underpressure distillation in Rotary Evaporators of the product after washing is removed to solvent and obtain 1-dodecyl-4-picoline Bromide, productive rate 94%.In round-bottomed flask, add 9.5mL phosphorus oxychloride, under ice-water bath cooling and stirring, drip 7.32mL DMF, drip and finish, in stirring at room 30min, slowly drip 25mL and contain 1 of 2.953g N-ethyl carbazole, 2-dichloroethane solution, drips and finishes, back flow reaction 8h.After having reacted, be cooled to after room temperature; reaction solution is poured in frozen water and stirred 1h, with dichloromethane extraction frozen water solution, then with P2O5, be dried the dichloromethane solution that contains product; methylene dichloride is removed in underpressure distillation afterwards, and product obtains 3-formyl radical-N-ethyl carbazole through column chromatography for separation.In the reaction flask of the 1-hexadecyl-4-picoline Bromide obtaining; add 1.004g3-formyl radical-N-ethyl carbazole; dehydrated alcohol 20.00mL, drips 3 piperidines, and back flow reaction 12h obtains red solution; after reaction solution is concentrated; add diethyl ether and separate out precipitation, suction filtration, filter cake is recrystallization in methyl alcohol; obtain required fluorescent probe, productive rate 30.7%.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 440nm, and emission wavelength is 566nm.As in Example 1, prepared fluorescent probe is configured to the probe solution that concentration is 0.020mmol/L, adopts inverted microscope to observe, can be observed bright microfilament bacterium mycelia under fluorescence condition, prepared probe can be identified microfilament bacterium.
Embodiment 4
Change the 0.5268g1-bromine n-Hexadecane in above-described embodiment 1 into 1.667g1-bromo-octadecane, obtain 1-octadecyl-4-picoline Bromide, productive rate 89.9%.Other operation, all by step in embodiment 3, does not adjust, and finally obtains the carbazole pyridine styrenic Cyanine Dyes Fluorescence probe that octadecyl chain is modified.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 435nm, and emission wavelength is 577nm.As in Example 1, prepared fluorescent probe is configured to the probe solution that concentration is 0.006mmol/L, adopts inverted microscope to observe, can be observed bright microfilament bacterium mycelia under fluorescence condition, prepared probe can be identified microfilament bacterium.
Embodiment 5
Change the 0.5268g1-bromine n-Hexadecane in above-described embodiment 1 into 1.4812g1-iodine dodecane, obtain 1-dodecyl-4-picoline iodized salt.Other operation, all by step in embodiment 3, does not adjust, and finally obtains the carbazole pyridine styrenic Cyanine Dyes Fluorescence probe that dodecyl chain is modified.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 441nm, and emission wavelength is 565nm.As in Example 1, prepared fluorescent probe is configured to the probe solution that concentration is 0.020mmol/L, adopts inverted microscope to observe, can be observed bright microfilament bacterium mycelia under fluorescence condition, prepared probe can be identified microfilament bacterium.
Embodiment 6
Change the 0.5268g1-bromine n-Hexadecane in above-described embodiment 1 into 1.7617g1-cetyl iodide, obtain 1-hexadecyl-4-picoline iodized salt.Other operation, all by step in embodiment 3, does not adjust, and finally obtains the carbazole pyridine styrenic Cyanine Dyes Fluorescence probe that hexadecyl chain is modified.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 436nm, and emission wavelength is 574nm.As in Example 1, prepared fluorescent probe is configured to the probe solution that concentration is 0.010mmol/L, adopts inverted microscope to observe, can be observed bright microfilament bacterium mycelia under fluorescence condition, prepared probe can be identified microfilament bacterium.
Embodiment 7
Change the 0.5268g1-bromine n-Hexadecane in above-described embodiment 1 into 1.902g1-iodine octadecane, obtain 1-octadecyl-4-picoline iodized salt.Other operation, all by step in embodiment 3, does not adjust, and finally obtains the carbazole pyridine styrenic Cyanine Dyes Fluorescence probe that octadecyl chain is modified.Fluorescent probe is carried out to spectral detection, and recording fluorescent probe maximum excitation wavelength is 443nm, and emission wavelength is 578nm.As in Example 1, prepared fluorescent probe is configured to the probe solution that concentration is 0.006mmol/L, adopts inverted microscope to observe, can be observed bright microfilament bacterium mycelia under fluorescence condition, prepared probe can be identified microfilament bacterium.
Claims (2)
1. identify the preparation method of fluorescent probe for microfilament bacterium for one kind; the method is take the synthetic of carbazole pyridine styrenic cyanine dyes and modify synchronized synthetic route; first introducing the long carbon chain compound of halo partly modifies its 4-picoline; then again with 3-formyl radical-N-ethyl carbazole partial reaction; make the fluorescent probe with long hydrophobic chain, comprise the following steps:
(1) chain alkyl-4-picoline halide salt is synthetic
In round-bottomed flask, add respectively 0.8-1.5 part 4-picoline, the long carbon chain compound of 0.7-1.3 part halo and 28 parts of toluene, magnetic agitation is fully dissolved and is reacted, temperature of reaction is 120 ℃, after question response completes, is cooled to room temperature, remove upper toluene and obtain oily matter, and with normal hexane washing, finally the oily matter having washed is carried out in Rotary Evaporators to underpressure distillation, obtain chain alkyl-4-picoline halide salt;
(2) 3-formyl radical-N-ethyl carbazole is synthetic
In round-bottomed flask, add 20 parts of phosphorus oxychloride, under ice-water bath cooling and stirring condition, in round-bottomed flask, drip successively DMF19-22 part, 65 parts containing 1 of 3-3.5 part N-ethyl carbazole, 2-dichloroethane solution, drips and finishes, back flow reaction 8h, reaction solution is cooled to room temperature, and pour in frozen water, stir, with dichloromethane extraction, in Rotary Evaporators, carry out after underpressure distillation, through column chromatography for separation, obtain 3-formyl radical-N-ethyl carbazole;
(3) fluorescent probe is synthetic
Chain alkyl-4-picoline halide salt of preparation in step (1) is dissolved in 70 parts of dehydrated alcohols; and add 3-formyl radical-N-ethyl carbazole of preparation in 0.9-1.3 part step (2); drip 3-5 and drip piperidines, back flow reaction 12h, obtains red solution; after reaction solution is concentrated; add ether to separate out precipitation, suction filtration, makes filter cake; by filter cake crystallization again in methyl alcohol, must there is the fluorescent probe of long hydrophobic chain.
2. identify according to claim 1 the preparation method of fluorescent probe for microfilament bacterium, it is characterized in that: the long carbon chain compound of described halo is 1-bromo-dodecane or 1-bromo-tetradecane or 1-bromine n-Hexadecane or 1-bromo-octadecane or 1-iodine dodecane or Cetyl Iodide or 1-iodine octadecane.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410225878.5A CN104017570B (en) | 2014-05-26 | 2014-05-26 | Identify the preparation method of microfilament bacterium fluorescent probe |
| PCT/CN2014/081349 WO2015180230A1 (en) | 2014-05-26 | 2014-07-01 | Method for preparing fluorescent probe for identifying microthrix parvicella |
| AU2014393669A AU2014393669B2 (en) | 2014-05-26 | 2014-07-01 | Preparation method of fluorescent probe for identification of Microthrix parvicella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410225878.5A CN104017570B (en) | 2014-05-26 | 2014-05-26 | Identify the preparation method of microfilament bacterium fluorescent probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104017570A true CN104017570A (en) | 2014-09-03 |
| CN104017570B CN104017570B (en) | 2016-01-20 |
Family
ID=51434582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410225878.5A Expired - Fee Related CN104017570B (en) | 2014-05-26 | 2014-05-26 | Identify the preparation method of microfilament bacterium fluorescent probe |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN104017570B (en) |
| AU (1) | AU2014393669B2 (en) |
| WO (1) | WO2015180230A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104479668A (en) * | 2014-10-15 | 2015-04-01 | 华中科技大学 | A fluorochrome probe |
| CN104845614A (en) * | 2015-04-30 | 2015-08-19 | 天津城建大学 | Fluorescent probe for marking DNA and synthetic method and using thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6579718B1 (en) * | 2000-08-04 | 2003-06-17 | Molecular Probes, Inc. | Carbazolylvinyl dye protein stains |
| CN101100465A (en) * | 2007-06-12 | 2008-01-09 | 山东大学 | Cation carbazole compound and application for the same as biphoton nucleic acid fluorescent probe |
| CN102743769A (en) * | 2011-04-21 | 2012-10-24 | 香港浸会大学 | Imaging beta-amyloid peptides and inhibition of beta-amyloid peptide aggregation |
| CN103087706A (en) * | 2013-01-18 | 2013-05-08 | 山东大学 | Single/double-photon acidic cell organelle fluorescent probe and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101234112B (en) * | 2008-03-03 | 2010-10-13 | 中国科学院化学研究所 | New purpose of cation carbazoles compounds |
| CN103333678B (en) * | 2013-06-25 | 2015-08-05 | 山东大学 | A kind of single, double photon line plastochondria fluorescent probe and application thereof |
-
2014
- 2014-05-26 CN CN201410225878.5A patent/CN104017570B/en not_active Expired - Fee Related
- 2014-07-01 AU AU2014393669A patent/AU2014393669B2/en active Active
- 2014-07-01 WO PCT/CN2014/081349 patent/WO2015180230A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6579718B1 (en) * | 2000-08-04 | 2003-06-17 | Molecular Probes, Inc. | Carbazolylvinyl dye protein stains |
| CN101100465A (en) * | 2007-06-12 | 2008-01-09 | 山东大学 | Cation carbazole compound and application for the same as biphoton nucleic acid fluorescent probe |
| CN102743769A (en) * | 2011-04-21 | 2012-10-24 | 香港浸会大学 | Imaging beta-amyloid peptides and inhibition of beta-amyloid peptide aggregation |
| CN103087706A (en) * | 2013-01-18 | 2013-05-08 | 山东大学 | Single/double-photon acidic cell organelle fluorescent probe and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| XIN JIANG FENG等: "Cyanines as New Fluorescent Probes for DNA Detection and Two-Photon Excited Bioimaging", 《ORGANIC LETTERS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104479668A (en) * | 2014-10-15 | 2015-04-01 | 华中科技大学 | A fluorochrome probe |
| CN104479668B (en) * | 2014-10-15 | 2016-08-31 | 华中科技大学 | A kind of fluorescence probe dye |
| CN104845614A (en) * | 2015-04-30 | 2015-08-19 | 天津城建大学 | Fluorescent probe for marking DNA and synthetic method and using thereof |
| CN104845614B (en) * | 2015-04-30 | 2016-07-13 | 天津城建大学 | A kind of DNA marker fluorescent probe and preparation method and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104017570B (en) | 2016-01-20 |
| AU2014393669A1 (en) | 2015-12-10 |
| WO2015180230A1 (en) | 2015-12-03 |
| AU2014393669B2 (en) | 2017-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Harrington et al. | Use of a fluorescent brightener to demonstrate cellulose in the cellular slime molds | |
| JP5237305B2 (en) | Detection and identification of microorganisms on permeable membranes | |
| CN107746382B (en) | 4-dansyl amide-indole fluorescent probe and preparation method and application thereof | |
| CN107502648A (en) | Unicellular drop high-throughput screening method based on micro-fluidic chip | |
| CN104830317A (en) | Sulfuretted hydrogen molecule fluorescent probe as well as preparation method and application thereof | |
| CN110156688B (en) | Fluorescent probe for detecting polarity of targeted endoplasmic reticulum and application thereof | |
| CN105504860B (en) | The synthesis and its application of a kind of pyranoquinoline fluorochrome | |
| CN103436608B (en) | Rapid detection method based on nucleic acid aptamers and kit | |
| CN104017570B (en) | Identify the preparation method of microfilament bacterium fluorescent probe | |
| CN110438252A (en) | Molecular labeling and its application with spinach male and female gender close linkage | |
| CN107760299B (en) | 6-dansyl amide-indole fluorescent probe and preparation method and application thereof | |
| WO2011033531A1 (en) | A reusable end-capped bipyridine compound useful for selective detection of cyanide anions and process for preparation thereof | |
| CN105296649B (en) | A kind of plant chromosome fluorescence in-situ hybridization method that improvement is simplified | |
| CN105754927A (en) | Preparation method of gracilaria salicornia protoplast | |
| CN104194379B (en) | A kind of thiazole orange class Molecule of Cyanine Dyes and application thereof | |
| EP2362890B1 (en) | Anthraquinone based near ir emitting compounds and uses thereof | |
| CN109022545A (en) | A kind of LAMP fluorescence visual colorimetric determination detection method and kit based on ruthenium complex | |
| CN104419763B (en) | Physical positioning method for cucumber single-copy gene on chromosome | |
| CN109913206A (en) | A kind of RNA fluorescence probe and its preparation method and application | |
| DE60014960T2 (en) | ALIZARIN DERIVATIVES AS NEW CHROMOGENIC SUBSTRATES | |
| CN114133413B (en) | Benzothiazole-triphenylamine compound and preparation method and application thereof | |
| CN106188014A (en) | The preparation of the c h bond Anion Recognition receptor of a kind of neutrality and application | |
| CN105968105B (en) | A kind of fluorescence labeling probe and preparation method thereof and the label application to bacterium | |
| CN105566206B (en) | The preparation and the application in terms of Heavy Metal Pollution Control of a kind of renewable fluorescence display material | |
| Vinodhini et al. | Assessment of antimicrobial property of a secondary metabolite produced by an enriched bacterial culture isolated from soil |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160120 |